A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases

Cherezov, Vadim; Peddi, A.; Muthusubramaniam, Lalitha; Zheng, Yuan F.; Caffrey, Martin

doi:10.1107/s0907444904019109

Cited by 139 publications

(154 citation statements)

References 7 publications

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“…Technologies for miniaturization of crystallization experiments are needed, but handling solutions of membrane proteins is complicated by their low surface tension and high viscosity. To address these challenges, both robotic (18,19) and microfluidic (20)(21)(22)(23) technologies have been developed to screen crystallization conditions on a submicroliter scale for both soluble and membrane proteins, but no universally applicable technology has yet emerged. Given the complexity of the problem of crystallization of membrane proteins, it is unlikely that a single technology would provide a universal solution, but there is a clear need for miniaturized technologies sufficiently robust, simple, and inexpensive to be accessible to individual laboratories.…”

mentioning

confidence: 99%

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

Li¹,

Mustafi²,

Fu³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

226

259

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High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a ''hybrid'' droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as Ϸ140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in Ϸ10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 l of protein solution, Ϸ1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.droplets ͉ plugs ͉ protein structure ͉ high-throughput ͉ miniaturization T his work reports a ''hybrid'' microfluidic approach that uses nanoliter plugs to perform screening and optimization simultaneously in the same experiment. To validate this method using a challenging problem, we demonstrate its compatibility with crystallization of membrane proteins. Small-scale screening and optimization experiments are important for biological assays, chemical screening, and protein crystallization (1-3). Screening and optimization are usually carried out sequentially. In the case of protein crystallization, random sparse matrix screening initially identifies the precipitants that may lead to crystallization. Subsequent gradient optimization establishes concentrations of these precipitants that lead to diffractionquality crystals (4). Combining screening and optimization steps into a single hybrid experiment would eliminate the need to wait for the outcome of the initial screen before carrying out subsequent optimizations. Furthermore, a hybrid experiment would reduce the false negatives (5) associated with screens performed at a single concentration. The hybrid experiment could also be more conclusive, because a single batch of the s...

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mentioning

confidence: 99%

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

Li¹,

Mustafi²,

Fu³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

226

259

View full text Add to dashboard Cite

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“…With the assistance of A. Peddi and Y. Zheng, engineers at The Ohio State University where the original work was carried out, we were able to perform a proof-of-principle robotics exercise employing LabViewcontrolled motorized translation stages operating and supporting a microsyringe and a crystallization plate. With it, we demonstrated that the viscous mesophase could be dispensed automatically and wells were filled in such a way that eventually yielded crystals [19]. This was enough to secure funding for a robot which was custom-designed and built to our specifications.…”

Section: The In Meso Robotmentioning

confidence: 91%

Crystallizing Membrane Proteins for Structure-Function Studies Using Lipidic Mesophases

Caffrey¹

2013

Advancing Methods for Biomolecular Crystallography

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The lipidic cubic phase method for crystallizing membrane proteins has posted some high-profile successes recently. This is especially true in the area of G-protein-coupled receptors, with six new crystallographic structures emerging in the last 3 1/2 years. Slowly, it is becoming an accepted method with a proven record and convincing generality. However, it is not a method that is used in every membrane structural biology laboratory and that is unfortunate. The reluctance in adopting it is attributable, in part, to the anticipated difficulties associated with handling the sticky viscous cubic mesophase in which crystals grow. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. However, over the years, we have worked to make the method user-friendly. To this end, tools for handling the mesophase in the pico-to nano-litre volume range have been developed for efficient crystallization screening in manual and robotic modes. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been implemented for a more rational approach to crystallogenesis, such that the method can now be applied to a wide variety of membrane protein types and sizes. In the present article, these assorted advances are outlined, along with a summary of the membrane proteins that have yielded to the method. The challenges that must be overcome to develop the method further are described.

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“…Most of the above obstacles are diminished by the development of robotic systems and microfocus beams. Robotic crystallization technologies, which can dispense subnanoliter-scale drops in 96-well plates within minutes, have expended the use of the lipidic cubic phase by substantially increasing the number of crystallization conditions that can be explored with limited amounts of sample [72,85] . Another recent innovation is the microfocus beamline at synchrotrons [86] , which makes data collection from the undersized GPCR crystals possible.…”

Section: Bicelle Methodsmentioning

confidence: 99%

Ice breaking in GPCR structural biology

Zhao

2012

Acta Pharmacol Sin

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G-protein-coupled receptors (GPCRs) are one of the most challenging targets in structural biology. To successfully solve a high-resolution GPCR structure, several experimental obstacles must be overcome, including expression, extraction, purification, and crystallization. As a result, there are only a handful of unique structures reported from this protein superfamily, which consists of over 800 members. In the past few years, however, there has been an increase in the amount of solved GPCR structures, and a few highimpact structures have been determined: the peptide receptor CXCR4, the agonist bound receptors, and the GPCR-G protein complex. The dramatic progress in GPCR structural studies is not due to the development of any single technique, but a combination of new techniques, new tools and new concepts. Here, we summarize the progress made for GPCR expression, purification, and crystallization, and we highlight the technical advances that will facilitate the future determination of GPCR structures.

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A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases

Cited by 139 publications

References 7 publications

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

Crystallizing Membrane Proteins for Structure-Function Studies Using Lipidic Mesophases

Ice breaking in GPCR structural biology

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